The purpose of this project will be to increase our understanding of the cellular mechanisms of glucocorticoid action on their target cells. The two aspects of this process to be investigated will be the mechanisms of glucocorticoid entry into the responsive AtT-20 mouse pituitary tumor cell and regulation of glucocorticoid binding sites in these cells. We will show whether glucocorticoid uptake is by a membrane transport process by examining the kinetics of glucocorticoid uptake into AtT-20 cell membrane vesicles. The estimation of free steroid influx will be easier since preparation of vesicles will make it possible to perform these studies in the absence of the intracellular receptor. Therefore we will use membrane vesicle to determine if a saturable, ligand-specific transport mechanism for glucocorticoids exists in these cells. The second aim of this project is to investigate our observation that exposure of AtT-20 cells to physiological concentrations of the natural glucocorticoid, corticosterone, leads to a rapid reduction in the total number of intracellular glucocorticoid binding sites. This project will determine if the binding site reduction is glucocorticoid-specific, and concentration-dependent. The effects of inhibitors of protein and RNA synthesis on binding site depletion and repletion will also be examined.